JP4531252B2 - Analytical method using particles and test kit for performing the method - Google Patents

Abstract

A method for use in a flow matrix, which utilizes biospecific affinity reactions to detect an analyte in the sample, and which comprises allowing the sample comprising the analyte and an analytically detectable reactant (Reactant*) to migrate through flow channels in a flow matrix to a detection zone located in the matrix, in which there is a firmly anchored biospecific affinity reactant (Capturer), and capturing the Reactant* in the detection zone in an amount related to the amount of analyte in the sample. The Reactant* has labeled particles of an analytically detectable group, and the Capturer is anchored to the matrix by immobilized particles which exhibit hydrophilic groups on their surface. A test kit comprises a flow matrix having a detection zone in which there is a firmly anchored biospecific affinity reactant (Capturer), and an analytically detectable reactant (Reactant*). The Reactant* has labeled particles of an analytically detectable group, and the Capturer is anchored to the matrix by immobilized particles which exhibit hydrophilic groups on their surface.

Description

【００６１】
【表２】

^*＝標識が結合しているときの、反応ゾーン内の吸光度(×１０００)。
【００６２】
結論
本実験は、結合粒子の形で保持した同じ量の樺アレルゲンが、アレルゲンを直接膜に保持させたものと比較して、樺特異的ＩｇＥ抗体の有意に高い結合を提供することを示す。
【００６３】
同様の実験において、異なる単分散ポリスチレン粒子(Bangs Laboratories)を固定粒子として、そして炭素粒子の代わりに使用し、異なる直径の蛍光ポリスチレンを使用した。固定粒子の直径は、０.２８−３μmの間で、異なる実験で変化した。標識粒子の直径は、０.１−０.５μmの間で、異なる実験で変化した。結果は、一般に、上記に詳述のような炭素粒子の結果に従う。
【００６４】
実施例２：アレルゲンが適用ゾーンに予め保持している試験変法での樺特異的ＩｇＥの測定
方法および材料
樺花粉アレルゲンのビオチニル化：ｔ３(樺花粉、Betula verrucosa)の抽出を、遠心および濾過溶液をPD-10カラムに付し、脱イオン水に溶出する以外、先に記載のように行った。ｔ３溶出物を凍結乾燥した(LSL SECFROID, LYOLAB F, ポンプ：LEYBOLD TRIVAC D8B)。
【００６５】
凍結乾燥ｔ３物質を０.１５Ｍ ＫＰＯ₄、０.１５Ｍ ＮａＣｌ、ｐＨ７.８に溶解した。含量の測定をアミノ酸分析により行った。本物質に、¹²⁵Ｉ−標識ｔ３を添加し、混合物を、２５mlの０.１５Ｍ ＫＰＯ₄、０.１５mＮａＣｌ、ｐＨ７.８で平衡化したPD-10カラムに適用した。ｔ３アレルゲンのビオチニル化を、供給者(Pierce)が推奨する条件にしたがって行った。３mgの溶出ｔ３抽出物(２.０ml)に、次いで、０.１３８mlのビオチン-LC-Sulfo-NHS(３.５９ｍＭ、Pierce)を添加し、インキュベーションをシェーカーで１時間、室温で行った。結合反応を５０μLの２Ｍグリシンの添加により停止させた。次いで、抽出物を、５０ｍＭ ＮａＰＯ₄、０.１５Ｍ ＮａＣｌ、ｐＨ７.４で平衡化したゲル濾過カラムに適用した。収率および最終タンパク質濃度は、得られた放射活性から測定した。
【００６６】
ストレプトアビジンのポリスチレン粒子への結合：
ストレプトアビジン(Molecular Probes)を、ＣＤＡＰ(１−シアノ−４−ジメチルアミノ−ピリジニウムブロミド)によりフェニルデキストラン吸着ポリスチレン粒子に結合させた(Kohn J and Wilechek M, FEBS Letters 154 (1) (1983) 209-210)。
【００６７】
ストレプトアビジンの脱塩および緩衝液交換を、ＮａＨＣＯ₃、０.１Ｍ、ｐＨ８.５中のゲル濾過(PD-10)により行った。３０％(容量)アセトン中の２％溶液の６００mgのフェニルデキストラン被覆ポリスチレン粒子を、４.５mlのCDAP(０.４４Ｍ)および３.６mlのＴＥＡ(０.２Ｍトリエチルアミン、Riedel-DeHaen)により活性化した。ＣＤＡＰを６０秒およびＴＥＡを１２０秒の撹拌で添加した。次いで、粒子を３０％(容量)アセトンで洗浄し、１２,１００ｇ(２５分、Beckman, J-21, JA-20, 10,000rpm)で遠心した。
【００６８】
２０.６mgのストレプトアビジンを３５０mgの活性化粒子に、シェーカーで１.５時間、＋４℃でインキュベーションすることにより結合させた。次いで、粒子を遠心し、その後不活性化を、ＮａＨＣＯ₃緩衝液中の０.０５Ｍグルタミン酸および０.０５Ｍアスパラギン酸で行った。インキュベーションを、一晩、＋４℃で撹拌しながら行った。結合粒子を、次いで、２回、５０ｍＭ ＮａＰＯ₄、０.０５％ＮａＮ₃、ｐＨ７.４で洗浄した。
粒子濃度は、分光測光法でＡ₆₀₀nmで、非処理粒子を対照として測定した。
【００６９】
ストレプトアビジン結合粒子のニトロセルロース膜への保持：ポリエステル裏打ちのあるニトロセルロースのシート(Whatman, ８μm、５cm幅)上に、１０ｍＭ ＮａＰＯ₄、５％スクロース、５％デキストラン５０００、ｐＨ７.４で１％粒子含量に希釈したストレプトアビジン結合粒子のゾーンを、Linear Striper (IVEK Corporation)で適用した。
保持流は２.５μl／cmおよび速度は２０mm／秒であった。保持物を１時間、３０℃で乾燥させ、その上で、シートを０.５cm幅切片に切った(Matrix 1201 Membrane Cutter, Kinematics Automation)。
【００７０】
濾紙へのビオチニル化アレルゲンの保持：１０×５mmフィルターを濾紙から切った(Whatman 3)。５０ｍＭリン酸緩衝液、ｐＨ７.４、ＢＳＡ６％中の１０μlのビオチニル化ｔ３(７７ng)をフィルターに分配し、フィルターを３０℃で４５分乾燥させた。
【００７１】
検出粒子への抗ｈＩｇＥ抗体の結合：ペプシンでfab'２フラグメントに開裂させたｈＩｇＥへの抗体を、表面にアルデヒド基を有する蛍光ポリスチレン粒子に結合させた(Molecular Probes C-17177 TransFluoSpheres, アルデヒド−スルフェートマイクロスフェア、０.１μm、６３３／７２０、２％固体)。２３mgの抗体を、５０ｍＭ ＮａＰＯ₄緩衝液、ｐＨ６中の６６mgの粒子に一晩、室温で結合させた。次いで、２０５μLのＮａＣＮＢＨ₄(５Ｍ)を添加して、結合を３時間、室温で減少させた。２０,８００×ｇ(Eppendorf 5417R中５０分、１４,０００rpm)で遠心後、不活性化を、脱イオン水、ｐＨ６.５中の０.０５Ｍグルタミン酸および０.０５Ｍアスパラギン酸で、一晩、室温で撹拌して行った。次いで、遠心を２０,８００×ｇ(５０分)で行った。０.０５％ＮａＮ₃含有５０ｍＭ ＮａＰＯ₄、ｐＨ７.４中の０.２％ＢＳＡでの遮断および一晩＋４℃でのインキュベーション後、遠心を再び２０,８００×ｇ(５０分)行った。２回の洗浄および遮断緩衝液中への貯蔵を次いで行った。粒子濃度を、螢光計(Perkin-Elmer LS50B)で、元々の粒子で調製した標準曲線により決定した。結合タンパク質濃度を、結合中に存在する放射活性抗ｈＩｇＥを有することにより測定した。
【００７２】
試験法：切片を、台盤平面から約１６°傾けて表面に重ねた。吸収膜(３.５cm幅、Schleicher & Schuell, GB004)を膜の上端から０.５cmに置いた。一定圧を得るために、金属重りを吸収膜上に置いた。次いで、サンプルおよび試薬を、連続的に下記に記載のようにピペット輸送した。各サンプルおよび試薬容量を次ぎの容量をピペット輸送する前に膜に重ねた。
【００７３】
１)３０μLの５０ｍＭ ＮａＰＯ₄、０.１５Ｍ ＮａＣｌ、ｐＨ７.４で予備洗浄。
２)予め保持させたビオチニル化ＩｇＥを有するフィルターを切片の底に置いた。
３)３０μLの血清を各フィルターにピペット輸送した。
４)２０μLの試験緩衝液(０.１ ＮａＰＯ₄、０.１５Ｍ ＮａＣｌ、１０％スクロース、３％ＢＳＡ、０.０５％ウシガンマグロブリン、０.０５％ＮａＮ₃、ｐＨ７.４)をフィルターに添加した。
５)アレルゲンフィルターを除去した。
６)試験緩衝液で希釈した２０μlの検出接合体(７５μg／mL)。
７)２×３０μLの試験緩衝液。
８)検出ゾーンの蛍光を、走査赤色レーザーフルオロメーター(６３５nm)で反応領域(Vmm)として測定した。
選択した血清サンプルは、陰性、弱い陽性および強い陽性血清を含んだ。
【００７４】
結果
【表３】

【００７５】
結果は、適用ゾーン中の予め保持させたアレルゲン(または抗原)および反応ゾーン中の一般的結合剤が十分機能することを示す。[0001]
Technical field
The present invention relates to a reagent (reactant) that is capable of analytically detecting a biospecific affinity reaction to measure an analyte in a sample. ^* ) And the method used in combination. The method involves the use of a liquid stream surrounded by a matrix that transports analytes and reactants to a detection zone (DZ) in / on the matrix. In the detection zone, there is a biospecific affinity reactant (capture) that is tightly immobilized on the matrix, and an amount of complex (reactant) that reflects the amount of analyte in the sample. ^* And in the detection zone). The invention also relates to a test kit for performing the method.
[0002]
A reactant (including an analyte) that exhibits biospecific affinity (bioaffinity reactant) and thus can be used in the present invention means an individual member of the reactant pair: antigen / hapten-antibody; biotin -Avidin / streptavidin; two complementary single strands of nucleic acid etc. For antibodies, Fab, F (ab) ₂ ', Antigen-binding antibody fragments such as single chain Fv (scFv) antibodies are contemplated. Related reactants can be naturally occurring as well as synthetically produced molecules / binders.
[0003]
The type of test method mainly uses biospecific affinity reactants in which at least part of the reactant pairs used exhibit protein structure, particularly in connection with so-called immunochemical assays.
Biospecific affinity reactions take place mainly in aqueous media (such as water).
[0004]
Previously used method
It is known in advance how to fix the catcher to the appropriate type of matrix. Alternatively, this is done via particles that are deposited in / on the matrix. The trap is then the reactant in the detection zone ^* It is bound to the particle through a bond that is stable under the conditions used for the capture. The bond between the capturer and the particle is generally a covalent bond, but physical and biospecific adsorption can also be used. See inter alia Abbott / Syntex US4,740,468; Abbott EP 472,476; Hybritech EP 437,287 and EP 200,739; Grace & Co. EP 420,053; Fuji Photo Film US 4,657,739; Boehringer Mannheim WO 94/06012. Reactants used for related types of tests ^* Suitable labeling groups are known, such as particles (Pharmacia AB WO 96/22532; Unilever WO 88/08534; Abbott Laboratories US 5,120,643, Becton Dickinson EP 284,232, etc.). The combination of particles as detectable groups and particles as fixed particles is also known from several of the above publications. See, for example, Boehringer Mannheim EP 462,376.
[0005]
Disadvantages of the prior art and objects of the present invention
There is often a need for improved detection sensitivity in connection with the previously known determination methods. A system that is often easy to manufacture is also desired.
The present invention aims to remedy these problems.
[0006]
The present invention
We prefer that the immobilization of the capture bodies via particles that are smaller than the smallest internal diameter of the flow channel in the flow matrix, but the reactant whose analytical indicator is a particle ^* And found to work surprisingly well together. Thus, the present invention provides:
A) An analytically detectable reactant (reactant) as a labeling group ^* ) Has particles, and B) the trap is itself anchored to the matrix through the particles, sized to be transported through the matrix and into the flow.
This is a test method characterized by the following.
[0007]
The particles present hydrophilic groups on their surface, preferably not belonging to the biospecific affinity reactant bound to the particles, especially if they are smaller than the matrix flow path. Preferred hydrophilic groups are uncharged (usually in the form of alcoholic hydroxyl groups).
As a rule, fixed particles are the reactants in the detection zone ^* If it is observed that it does not interfere with the detection of the labeled particles and the immobilized particles can be of the same type.
[0008]
The particles intended to immobilize the trap on the DZ are preferably smaller than the minimum internal diameter of the flow path, as described above. Suitable particle sizes (maximum external size / diameter) are in the range of 0.1-1000 μm, preferably 0.1-100 μm. Each example relating to the minimum internal diameter of the flow path of the matrix used must be considered. The particles used can be polydisperse or monodisperse. Its shape can vary from spherical to completely staggered. Suitable particulate materials that can be mentioned in particular are, for example, SiO. ₂ and
(A) Synthetic polymers such as condensation polymers and addition polymers. Among the addition polymers, mention may be made of alkyl vinyl ethers, aryl vinyl ethers, vinyl allenes (such as styrene and divinyl benzene), alkyl alkenes, acrylates, methacrylates, acrylamides, methacrylamides, etc., and
(b) optionally synthetically cross-linked (e.g. semi-synthetic polymers) biopolymers such as polysaccharides (agarose, dextran, starch), etc.
Other polymeric materials such as organic polymers selected from.
[0009]
In this connection, so-called latex particles, often polymerized styrene or other polymerized alkenes / alkadienes, are often used. The fixed particles can be porous or non-porous.
[0010]
The choice of fixed particles that are moderate in terms of hydrophobic and hydrophilic properties is often important. The reason is that although the flow matrices often exhibit significant hydrophobicity, they are sufficiently hydrophilic to flow in an aqueous liquid medium. For example, the highly hydrophobic particles of polystyrene thus adsorb very strongly on the nitrocellulose membrane. The same is also true for other flow matrices where a balance between hydrophilic and hydrophobic properties is in place. Unfortunately, the hydrophobic nature of the particles ^* And / or promote non-specific adsorption of analytes. This reduces the sensitivity of the test method. In our system we have therefore chosen to hydrophilize hydrophobic particles, for example by inserting hydrophilic groups such as hydroxy groups on their surface. It is particularly convenient to coat the hydrophobic particles with polyhydroxy polymers or other hydrophilic polymers, preferably substituted with hydrophobic groups, for example hydrocarbyl groups such as phenyl. Examples of hydrocarbyl-substituted hydrophilic polymers that can be used are those having a polysaccharide structure, such as phenyldextran. The presence of hydrophobic groups on the hydrophilic polymer facilitates adsorption of the polymer to the hydrophobic particles. This in turn reduces the need for stabilization of the adsorbed polymer via cross-linking. In production engineering, especially for small sized particles often used in connection with the present invention, crosslinking can be very important as it easily leads to particle agglomeration. Insertion of a hydrophobic group into the particle means that covalent binding of the biospecific affinity reactant to the particle can be more easily achieved. Hydrophilization itself also reduces the tendency for non-specific adsorption in the detection zone.
[0011]
Detectable reactant ^* The particles intended for are usually smaller than those used for fixation. Suitable particle diameters are usually selected from 0.001-5 μm, often preferably from colloidal features, so-called sols (ie having a size between 0.001-1 μm, usually spherical and monodisperse) ). In principle, the same particulate material as the fixed particles can be used. Known label particles include metal particles (e.g. gold sol), non-metal particles (SiO ₂ Carbon, latex (polystyrene) and killed red blood cells and bacteria). With respect to particles with non-colloidal properties, it is true that they should not be precipitated under conditions effective for matrix transport. Therefore, more or less complete and more or less polydispersed carbon particles (<1 μm) have been used (Pharmacia AB, WO 96/22532). The particles can be provided with groups that facilitate their detection, for example by providing chromophores, fluorophores, radioactive compounds, enzymes, and the like. In the present invention, it has been shown that fluorescent particles rather than colored particles, such as carbon particles, are surprisingly advantageous.
[0012]
The requirements for the equilibrium between the hydrophobic and hydrophilic properties of the labeled particles are similar to those that apply for fixed particles.
[0013]
When the trap with fixed particles is held in the detection zone, it is essential to select conditions that promote physical adsorption to the matrix. Drying is often essential. Once the bonds between the matrix and the fixed particles are formed, it is often difficult to break them. But the reactant ^* Should be applied under conditions that promote the reactants to be maintained in suspension and do not promote physical adsorption of the particles to the matrix. Reactant ^* Is pre-retained in the matrix, it is essential to do so in a manner that facilitates rapid resuspension for transport to the matrix. Below “Bio-specific affinity reactant application zone other than specimen (A _R Z) ”.
[0014]
In the detection zone, the analyte can bind to the capture body directly or indirectly. In the latter case, the capture body is a biospecific affinity reactant that can bind to additional reactants that can bind to the analyte via biospecific affinity. In this case, this further reactant does not have to be fixed to the matrix from the beginning, but is pre-held mobilized (diffusible) in the area away from the detection zone or in the matrix within the zone, or together with the sample. Or can be added separately. When this further reactant is in a soluble form, the capture body is advantageously one member of a specific binding pair, and the other member binds or joins the reactant. Examples of such specific binding pairs are immunological binding pairs such as antigen-antibody and hapten-antibody, biotin-avidin or -streptavidin, lectin-sugar, nucleic acid duplex.
[0015]
The particle system of the invention is particularly advantageous in allergy tests where the allergen that reacts with the analyte (most often the IgE class) is usually a complex mixture of 100 or more proteins. Binding of the protein to the particle and its pre-retention results in a very strong immobilized allergen that does not selectively leak most of the components compared to allergens that are passively adsorbed to the matrix. This, combined with the fact that particle labels provide a very good signal, provides a special test system for allergies. The above applies to all tests in which complex binding agents, such as autoantigens in the determination of autoimmune diseases, are used.
[0016]
Variants of soluble reactants (allergens) that have been pre-held or added with the sample, on the one hand, have a rather long incubation time between the particle label and the allergen / analyte, while the soluble allergen has a solid phase with the allergen. Other advantages may be provided in allergy testing because it is available by reaction with the analyte rather than when bound to.
[0017]
matrix
The matrix defines a space in which the reactants are transported. The matrix can be the inner surface of a single channel (eg, a capillary), and the inner surface of the porous matrix with the channel system extends through it. This type of matrix is referred to below as a flow matrix. The flow matrix may exist in the form of a monolith, sheet, column, membrane, single channel having capillary dimensions, or an integrated system of such channels. They can also be present in column packaging, in the form of particles packed in compressed fibers, and the like. The inner surface of the matrix is hydrophilic and an aqueous medium (usually water) can be absorbed into the matrix and transported. The minimum internal diameter of the channel must be large enough for the reactants used to be transported through the matrix. The definition of the knob is that if the matrix has a system of interrelated channels, a suitable matrix is selected from those having a minimum internal diameter channel between 0.4 and 1000 μm, preferably between 0.4 and 100 μm. Can be selected. A channel with a minimum internal dimension at the top with a wide spacing (up to 1000 μm) is mainly interesting when the flow is driven by external pressure / suction.
[0018]
The target matrix is constructed from a polymer such as nitrocellulose, nylon, and the like. The material of the matrix, as well as the physical and geometric design of the flow path, can vary along the flow and depends on which specific part of the matrix is used (Pharmacia AB WO96 / 22532; Medix WO94 / 15215).
[0019]
Along the transport flow in the matrix, the sample (A _S Z), reactant (A _R Z), buffer solution (A _B There may be zones for applications such as Z), and zones for detection (DZ) and calibrator (CZ, below).
[0020]
Various flow matrices that can be used in particular test types have been described in previous patent publications (Behringwerke US 4,861,711, Unilever WO 88/08534, Abbott US 5,120,643 and US 4,740,468; Becton Dickinson EP 284,232 and US 4,855,240; Pharmacia AB WO 96/22532 etc.).
[0021]
Transport flow
The direction of the transport flow is from the application zone to the detection zone (DZ). The exact zone through which the transport stream passes is determined by the particular test protocol. The transport stream spreads radially and can start from a point or part of the flow tip in the form of a circular perimeter. The transport stream can also start from a band-shaped zone and can have a straight flow tip perpendicular to the direction of flow.
[0022]
In a less preferred variant, the transport stream advances from the sample application zone, which is also the detection zone. In this variant, the flow preferably extends from the application / detection zone, preferably radially, and possibly through a further downstream detection zone.
[0023]
Transport flow through a particular type of matrix can be achieved by the action of capillary forces, for example by starting with a substantially dry matrix. The absorber may be placed at the extreme end of the flow for assistance. A flow that is primarily considered for the transport of dissolved components can be achieved when the electric field is loaded across the matrix (flow direction).
The flow utilized is preferably sideways, i.e. parallel to the upper surface of the matrix. Other types of flows (back of the matrix) can also be used.
[0024]
Related testing protocols
The present invention applies primarily to non-competitive (non-inhibitory) test variants, but can also be applied to competitive (inhibition) test variants. The complexes formed with different test protocols are shown schematically below. The relevant reactant is assumed to be monovalent with respect to the binding site utilized. The protocol can be run as a simultaneous or sequential variation with respect to the analyte and the loading reactant. Co-variant means that the analyte (sample) and the reactants are co-transported at least partly during transport and preferably reach the detection zone simultaneously. Continuous variant means that the analyte (sample) travels in front of the reactant, at least part of the transport towards the detection zone, and preferably arrives in front of the reactant in the detection zone. The test protocol of the present invention comprises a specimen and a reactant. ^* Are always simultaneous or sequential. “-” Relates to a rigid fixation from the beginning. “---” relates to binding via biospecific affinity.
[0025]
A. Sandwich protocol:
Capturers and reactants ^* Has a biospecific affinity for the analyte. x is the number of moles of traps on the matrix. y is the number of moles of analyte bound to the capture body (= reactant) ^* Mole).
Complex formed in the detection zone:
Matrix (-capturing body) _xy (-Capture --- Specimen --- Reactant ^* ) _y .
[0026]
B. Sandwich protocol:
The capturer has a biospecific affinity for reactant I, which in turn has a biospecific affinity for the analyte. Reactant ^* Has a biospecific affinity for the analyte. x is the number of moles of traps on the matrix. y is the number of moles of analyte bound to the capture body via reactant I (= reactant ^* Mole). y + z is the number of moles of reactant I bound to the trap.
Complex formed in the detection zone:
Matrix (-capturing body) _xzy (-Capture --- reactant I) _z (-Capture --- reactant I --- analyte --- reactant ^* ) _y .
[0027]
C. Inhibition protocol:
The capture body is an analyte analog and has a binding site equivalent to the binding site of the analyte. Reactant II has a biospecific affinity for the analyte and capture body. Reactant ^* Has a biospecific affinity for reactant II. x is the number of moles of traps on the matrix. y is the number of moles of reactant II bound to the matrix via the trap (= reactant ^* Number of moles). Reactant II is part of the complex in an amount proportional to the amount of analyte in the sample.
Complex formed in the detection zone:
Matrix (-capturing body) _xy (-Capturer --- Reactor II --- Reactor ^* ) _y .
[0028]
D. Inhibition protocol:
Capture bodies are specimens and reactants ^* Both show biospecific affinities. Reactant ^* Is a detectable soluble analyte analog. x and y are each a reactant bound to the matrix via a trap ^* And the number of moles of specimen. x + y is the number of moles of trap on the matrix.
Complex formed in the detection zone:
Matrix (-capture --- reactant ^* ) _x (-Capture --- specimen) _y .
[0029]
Sample application zone (A _s Z)
This type of zone should be seen upstream of the intended analyte detection zone.
[0030]
Non-specimen biospecific affinity reactant application zone (A _R Z)
The order of application zones depends on the test protocol and specimen and reactant. ^* It should be ensured that they are simultaneous or sequential. This is the reactant ^* (A _{R *} Z) for the reactant application zone (A _R Z) means that it must always be upstream of the detection zone. One or more reactants may be added to the same application zone. The application zone is the sample and at least one reactant (= A _R Z / A _S Z), for example, reactant ^* (= A _{R *} Z / A _S If common in Z), the applications are made simultaneously, for example, mixing the sample and reactants before providing them to the zone. If desired, the mixture can be preincubated so that the reactants bind to the analyte or other component in the intended manner prior to application. With knowledge of the various protocols, one skilled in the art can easily determine which zones are required and in what order they should be located.
[0031]
The reactants used in the method can be pre-held in their respective zones or added in connection with the performance of the measurement method. Pre-holding involves application of the reactants in advance in such a way that it does not spread outside the application zone until the liquid flow begins.
[0032]
Pre-retention of the reactants can be done in a manner known per se (see for example Behringwerke US 4,861,711; Unilever WO 88/08534; Abbott US 5,120,643; Becton Dickinson EP284,232). It is important to consider the fact that when the liquid reaches a previously held reactant, it must dissolve. In order to achieve fast dissolution, it is common to include a related half-single in a material that dissolves faster upon contact with the liquid medium used. This type of material is often hydrophilic with polar and / or charged groups such as hydroxy, carboxy, amino, sulfonate and the like. In particular, hydrophilic fast-dissolving polymers may be mentioned, for example having carbohydrate structures, simple sugars including mono-, di- and oligosaccharides and the corresponding sugar alcohols (mannitol, sorbitol etc.). It is common practice to first coat the application zone with a layer of fast-dissolving material and optionally apply the reactants following the addition of a layer of further fast-dissolving material. An alternative is by including the reactants in particles of a material that dissolves quickly and then retaining them in the relevant zone of the matrix.
[0033]
Buffer zone (A _B Z)
The required buffer system can be included in the solution added simultaneously with the sample and reactants. In traditional methods, the addition of buffer is done in an application zone located upstream of all other application zones. This is often the same as the sample application zone. In the present invention, the buffer can be added at a desired position (see below).
[0034]
In the simultaneous PCT application “Analytical method including addition at two or more positions and its apparatus and test kit” (based on SE9704934-0), we provide that one variant provides a preferred embodiment of the invention This invention is described. This patent application is incorporated herein by reference. The invention in this other patent application is derived from two continuous zones (AZ2 and AZ1) in the flow matrix if the liquid is applied to the downstream zone (AZ1) at the same time or before the liquid is applied to the upstream zone. Based on the discovery that the liquids can move after each other without mixing. This discovery was guided by the ability to achieve zone-wise movement of any reactant present in the liquid toward the detection zone. Sample application zone (A _S Z) is the reactant ^* Applicable zone (A _{R *} When located downstream of Z), the test protocol is ^* At the same time. A _{R *} Z and A _S One zone for application of liquid alone (buffer) during Z (A _B Z) is present, the detection zone DZ is washed away by the analyte and reactant. ^* Made during the capture. Such an intermediate buffer zone (A _B Z) may ensure that reactants (including analytes) that apply to the zone located downstream start from the application zone located upstream and arrive at the DZ before the reactants. The latter can be important when delaying the reactants being applied to the zone where the matrix itself is located downstream.
[0035]
The reactants can be included in the liquid applied to the zone. Alternatively, with respect to the application of the liquid, it can be held in advance in the zone to which the corresponding liquid is applied or in the zone located between this and the nearest downstream zone.
[0036]
This alternative invention allows the application of the buffer in the present invention to be performed at a desired location. According to conventional practice, addition of buffer is possible only in the application zone upstream of other application zones.
This aspect of the invention is directed to reactants ^* Especially interesting in a way that is continuous with respect to.
[0037]
Specimen
The present invention is primarily suitable for the determination of biospecific affinity reactants of the type described at the outset. The specimen can be a cell or virus or part thereof. Special mention may be made of antigens such as immunoglobulins or antibodies. For immunoglobulins, the measurement may be for a certain Ig and / or a certain Ig subclass. With respect to antibodies, the measurement can relate to a specific antibody, optionally also the Ig class or Ig subclass of the antibody. Related Ig classes are IgA, IgD, IgE, IgG and IgM. Related Ig subclasses are IgG1, IgG2, IgG3 and IgG4.
[0038]
In a variant of the sandwich (according to protocols A and B above), the specimen is an antibody directed against allergen / antigen / hapten and is derived from a specific species, a specific Ig class or a specific Ig subclass. In this case, the reactant ^* Are directed to an epitope that is specific for a species, Ig class or Ig subclass by capturer (protocol A) and reactant I (protocol B) and allergen / antigen / hapten. Alternatively, the reverse is also selected, ie capturer and reactant I are each antibodies directed to the analyte. When the specimen is generally an antigen, for protocol A, the capture and reactant ^* Can be antibodies directed against both antigens. For protocol B, it is the reactant I and the reactant directed to the antigen. ^* It is.
[0039]
Competitive variants are most interesting mainly for low molecular weight analytes. Illustrative examples are antigens and haptens. In protocol C, the capture body is an antigen or hapten tightly bound to the matrix, reactant II is an antibody directed to the antigen, and the reactant ^* Is an antibody directed to Reactant II. In protocol D, the capture bodies are antibodies and reactants directed to the specimen. ^* Can be an analyte labeled with an analytically detectable group.
[0040]
The methods of the invention can be performed as part of a diagnosis of an allergy or an autoimmune disease.
Measurement of IgE or IgG class anti-allergen antibodies is of particular interest to the inventor, and in the latter case, the emphasis is preferably on some of the described subclasses. Measurement of allergen-specific antibodies can be used in connection with the diagnosis of IgE-mediated allergies.
[0041]
As already mentioned above, the present invention relates to the case where the capture body consists of a mixture of different components, for example an allergen, often consisting of a mixture of several different allergenic components, and the analyte is directed to the individual components of the mixture. Particularly suitable when the antibody is a modified antibody.
[0042]
sample
Suitable samples are of biological origin, e.g. different body fluids (whole blood, serum, plasma, urine, tears, spinal fluid, etc.), cell culture media, processing methods in biotechnology, food, environment (environmental analysis samples) Or the like. The sample can be pretreated to fit, for example, a matrix, the test protocol involved, etc.
[0043]
Calibrator
The type of measurement method involving the present invention involves analytically detectable reactants (reactants). ^* ), And the measurement signal (sample value) is taken as a scale for the analyte in the sample. In order to transfer the measurement signal to the actual sample amount, the signal is compared with the corresponding signal (calibrator value) of a known standard amount of sample (calibrator). In connection with the present invention, a new calibrator system has been developed to apply the structure of the present invention in the best mode.
[0044]
This further invention means that the calibrator is pre-fixed (matrix calibrator), preferably in the same type of matrix used for the sample run. When measuring the calibrator value, the matrix calibrator ^* And then the reactants ^* Is measured in a manner known per se. By using different amounts of matrix calibrator, a series of calibrator values can be obtained corresponding to different predetermined amounts of analyte in the sample (standard amount, dose response curve, calibration curve).
[0045]
Instead of pre-fixing the calibrator to the matrix, the reactants that can bind to the calibrator can be fixed and then the calibrator can be added in connection with the measurement of the calibrator value. As the calibrator binding agent binds to the matrix, the calibrator may be pre-held in a zone (region) away from the detection zone (movable) or added with the sample or separately.
[0046]
The application of the present invention to our new calibrator system relates to the transport stream passing through one or more zones with calibrators firmly fixed to the matrix in each calibrator zone (KZ).
[0047]
Binding of the calibrator or calibrator binder to the matrix in the calibrator zone can be done on the same principle as the immobilization of reactant I to the detection zone. The calibrator binding agent is usually one of the members of a specific binding pair (reactant pair), and the other member of the binding pair is bound to or conjugated to the calibrator substance. Examples of such specific binding pairs are described above in connection with the description of the capture body.
[0048]
Calibrator zone is the reactant ^* Should be located downstream of the liquid application zone, intended for transport of the liquid. In relation to the detection zone (DZ), the calibrator zone is preferably located upstream.
Our invention related to calibrators is detailed in a co-pending PCT application entitled “Use of New Calibrators and Equipment and Test Kits Containing the Calibrators” (based on SE9704933-2). This application is incorporated herein by reference.
[0049]
Second main aspect of the present invention
This aspect of the invention comprises (a) an analytically detectable biospecific affinity reactant (reactant) wherein the labeling group is a particle. ^* ) With a flow matrix having a detection zone in which (b) the capture body is rigidly fixed via particles, preferably smaller than the smallest diameter of the flow path. The associated particles and channels follow those described above. The flow matrix may show application zones, pre-retained reactants, etc. according to the above.
The invention is described in the Examples section and is defined in the claims.
[0050]
Example
Example 1: Comparison of birch allergens bound to the detection zone via particles or adsorbed directly to the detection zone
This example is based on the measurement of IgE specific for sputum allergens. To demonstrate the strength of the present invention, the responses obtained from many patient samples were: 1) the allergen extract was covalently bound to polystyrene particles coated with phenyldextran retained in the detection zone. And 2) a test variant in which the allergen extract is directly retained and passively bound to the nitrocellulose membrane.
[0051]
Methods and materials
Adsorption of phenyldextran on polystyrene particles: phenyldextran dissolved in dow ion water at various concentrations (substitution degree: 1 phenyl group per 5 monosaccharide units = 20%, Mw dextran 40,000, Pharmacia Biotech AB Uppsala, Sweden) with polystyrene particles (0.49 μm, Bangs Laboratories, USA): 1) 4-5 mg / ml, 8-10% particle suspension, RT 1h, 2) 5 mg / ml, 5% particle suspension It was adsorbed by stirring overnight with a suspension, RT 1h, 3) 20 mg / ml, 2% particle suspension. The particles were subsequently washed twice with deionized water. The particle suspension was centrifuged and washed for each incubation (12,100 × g, 30 minutes, Beckman, J2-21).
[0052]
Extraction of t3 (Betula verrucosa): 1 part (weight) of camellia pollen (Allergon, Sweden) was extracted with 10 parts (volume) of 0.1 M phosphate buffer, pH 7.4. Extraction was continued for 2 hours at + 4 ° C. on a shaker desk (200 pulses / min). The extract was centrifuged at 4000 rpm for 1.75 hours. After filtration, the t3 extract was applied to a PD-10 column (Pharmacia Biotech AB, Sweden) and 0.1M NaHCO 3. _Three Elute at pH 8.5. The t3 eluate (nomenclature: t3 extract 1/14) was taken for analysis in the reservoir for all levels of protein.
[0053]
Binding of t3 extract to polystyrene particles (t3 particles): t3 extract was converted to CDAP (1-cyano-4-dimethylamino-pyridinium bromide) (Kohn J and Wilchek M, FEBS Letters, 154 (1) (1983) 209 -210) to phenyl dextran coated polystyrene. A 2% particle suspension of polystyrene particles (2128 mg) coated with phenyldextran in 30% (volume) acetone was mixed with 954 mg CDAP (100 mg / ml in 30% acetone) and 7.63 ml 0.2M triethylamine (TEA, Riedel). -de Haen, Germany). CDAP was added with stirring, TEA was added dropwise for 90 seconds and stirred for a total of 120 seconds. The reaction was stopped by adding 30% acetone (4 volumes) and centrifuged at 12,400 × g for 35 minutes. The particles were washed once with deionized water.
[0054]
0.1M NaHCO _Three 640 ml of t3 extract 1/14 in pH 8.5 was added to the activated particles and the binding reaction was continued on a shaker machine for 1 hour. The suspension is spun down and then 0.1M NaHCO. _Three Inactivated with 0.05M aspartic acid and 0.05M glutamic acid in pH 8.5. Incubated overnight at + 4 ° C. on a shaker desk. Particles are 50 mM NaPO _Four 0.05% NaN _Three Washed by centrifugation in pH 7.4.
[0055]
The concentration of particles was measured spectrophotometrically at 600 nm with untreated particles as a control. t3-linked polystyrene particles were subjected to amino acid analysis for determination of total protein levels.
[0056]
Retention of t3 extract and t3 particles to membrane (detection zone): on a polyester-lined sheet of nitrocellulose (Whatman, 8 μm, 5 cm width), 1/3 of t3 extract 1 μl / s and 1 μl / s This was applied by Linear Striper (IVEK Corporation). t3 extract 1/14 undiluted and 0.1M NaHCO _Three , Maintained at a 1: 1 dilution of pH 8.5 (t3 extract 1/28). t3 particles were mixed with 50 mM NaPO _Four 6% lactose, 0.05% NaN _Three Dilute to 4% particle level at pH 7.4. The sheet containing the retentate was dried at 30 ° C. for 1 hour. Sheets were cut into 0.5 cm sections (Matrix 1201 Membrane Cutter, Kinematics Automation).
[0057]
Carbon particle assembly (reactant) ^* ): Monoclonal anti-human IgE antibody (anti-hIgE) was adsorbed onto carbon particles (sp100, <1 μm, Degussa, Germany) according to WO 96/22532. The final suspension diluted with test buffer contained 300 μl / ml carbon particles.
[0058]
Test method: The slices were tilted about 16 ° from the platen plane and overlapped on the surface. Overlaid membranes (3 cm wide, Whatman, 17 Chr) were placed 0.5 cm at the end of the section. In order to obtain a constant pressure, a metal weight was placed on the absorbent membrane.
[0059]
Samples and reagents were pipetted in the following order. Each sample and reagent volume was moved into the membrane before pipetting the subsequent volume.
1) 30 μl test buffer (0.1 M Tris-HCl, 0.6 M NaCl, 10% sucrose, 3% bovine serum albumin, 0.05% bovine gamma globulin, pH 7.4)
2) 30 μl serum sample
3) 20 μl of test buffer (same as step 1)
4) 20 μl of carbon particle conjugate (anti-hIgE antibody adsorbed on carbon particles, 300 μg / ml, diluted with test buffer)
5) 2 × 30 μl test buffer
6) Carbon blackening in the reaction zone was measured as absorbance with an Ultroscan XL, Enhanced Laser Densitometer (LKB).
[0060]
result
The amount of protein in the detection zone
[Table 1]

[0061]
[Table 2]

^* = Absorbance in the reaction zone when the label is bound (x1000).
[0062]
Conclusion
This experiment shows that the same amount of sputum allergen retained in the form of bound particles provides significantly higher binding of sputum-specific IgE antibodies compared to those with the allergen retained directly on the membrane.
[0063]
In a similar experiment, different monodisperse polystyrene particles (Bangs Laboratories) were used as fixed particles and instead of carbon particles, and different diameters of fluorescent polystyrene were used. The diameter of the fixed particles varied between 0.28-3 μm in different experiments. The diameter of the labeled particles varied between 0.1 and 0.5 μm in different experiments. The results generally follow the results for carbon particles as detailed above.
[0064]
Example 2: Measurement of sputum-specific IgE in a test variant in which the allergen is pre-retained in the application zone
Methods and materials
Biotinylation of camellia pollen allergen: Extraction of t3 (bamboo pollen, Betula verrucosa) was performed as described above except centrifuge and filtered solution applied to a PD-10 column and eluted in deionized water. The t3 eluate was lyophilized (LSL SECFROID, LYOLAB F, pump: LEYBOLD TRIVAC D8B).
[0065]
Lyophilized t3 material with 0.15M KPO _Four , 0.15 M NaCl, pH 7.8. The content was measured by amino acid analysis. To this substance ¹²⁵ I-labeled t3 is added and the mixture is taken up with 25 ml of 0.15 M KPO. _Four , Applied to a PD-10 column equilibrated with 0.15 mM NaCl, pH 7.8. Biotinylation of the t3 allergen was performed according to conditions recommended by the supplier (Pierce). To 3 mg of eluted t3 extract (2.0 ml) was then added 0.138 ml of biotin-LC-Sulfo-NHS (3.59 mM, Pierce) and incubation was performed on a shaker for 1 hour at room temperature. The binding reaction was stopped by the addition of 50 μL of 2M glycine. The extract was then washed with 50 mM NaPO. _Four , Applied to a gel filtration column equilibrated with 0.15 M NaCl, pH 7.4. Yield and final protein concentration were determined from the radioactivity obtained.
[0066]
Binding of streptavidin to polystyrene particles:
Streptavidin (Molecular Probes) was coupled to phenyldextran-adsorbed polystyrene particles by CDAP (1-cyano-4-dimethylamino-pyridinium bromide) (Kohn J and Wilechek M, FEBS Letters 154 (1) (1983) 209- 210).
[0067]
Streptavidin desalting and buffer exchange were performed using NaHCO 3. _Three Gel filtration (PD-10) in 0.1M, pH 8.5. Activation of 600 mg phenyldextran coated polystyrene particles in 2% solution in 30% (volume) acetone with 4.5 ml CDAP (0.44 M) and 3.6 ml TEA (0.2 M triethylamine, Riedel-DeHaen) did. CDAP was added for 60 seconds and TEA for 120 seconds with stirring. The particles were then washed with 30% (volume) acetone and centrifuged at 12,100 g (25 minutes, Beckman, J-21, JA-20, 10,000 rpm).
[0068]
20.6 mg of streptavidin was coupled to 350 mg of activated particles by incubation at + 4 ° C. for 1.5 hours on a shaker. The particles are then centrifuged and then inactivated by NaHCO 3 _Three Performed with 0.05 M glutamic acid and 0.05 M aspartic acid in buffer. Incubation was performed overnight at + 4 ° C. with agitation. The bound particles are then washed twice with 50 mM NaPO _Four 0.05% NaN _Three And washed at pH 7.4.
The particle concentration is determined by spectrophotometry. ₆₀₀ At nm, untreated particles were measured as a control.
[0069]
Retention of streptavidin-bound particles on nitrocellulose membrane: on a nitrocellulose sheet (Whatman, 8 μm, 5 cm width) with polyester backing, 10 mM NaPO _Four A zone of streptavidin-bound particles diluted to 1% particle content with 5% sucrose, 5% dextran 5000, pH 7.4 was applied with a Linear Striper (IVEK Corporation).
The holding flow was 2.5 μl / cm and the speed was 20 mm / sec. The retentate was dried for 1 hour at 30 ° C., after which the sheet was cut into 0.5 cm wide sections (Matrix 1201 Membrane Cutter, Kinematics Automation).
[0070]
Retention of biotinylated allergen on filter paper: A 10 x 5 mm filter was cut from the filter paper (Whatman 3). 10 μl of biotinylated t3 (77 ng) in 50 mM phosphate buffer, pH 7.4, 6% BSA was dispensed onto the filter and the filter was dried at 30 ° C. for 45 minutes.
[0071]
Binding of anti-hIgE antibody to detection particles: An antibody to hIgE cleaved with pepsin into a fab'2 fragment was bound to fluorescent polystyrene particles having an aldehyde group on the surface (Molecular Probes C-17177 TransFluoSpheres, Aldehyde-Sulfurs). Fate microspheres, 0.1 μm, 633/720, 2% solids). 23 mg of antibody was added to 50 mM NaPO _Four Bound to 66 mg particles in buffer, pH 6, overnight at room temperature. Then 205 μL NaCNBH _Four (5M) was added to reduce binding for 3 hours at room temperature. After centrifugation at 20,800 × g (50 minutes in Eppendorf 5417R, 14,000 rpm), the inactivation was performed at room temperature overnight with 0.05 M glutamic acid and 0.05 M aspartic acid in deionized water, pH 6.5. And stirring. Centrifugation was then performed at 20,800 × g (50 minutes). 0.05% NaN _Three Contains 50 mM NaPO _Four After blocking with 0.2% BSA in pH 7.4 and incubation at + 4 ° C. overnight, centrifugation was again performed at 20,800 × g (50 minutes). Two washes and storage in blocking buffer were then performed. The particle concentration was determined with a fluorometer (Perkin-Elmer LS50B) according to a standard curve prepared with the original particles. The bound protein concentration was determined by having radioactive anti-hIgE present during binding.
[0072]
Test method: Sections were tilted approximately 16 ° from the platen plane and overlaid on the surface. An absorbent membrane (3.5 cm wide, Schleicher & Schuell, GB004) was placed 0.5 cm from the top of the membrane. In order to obtain a constant pressure, a metal weight was placed on the absorbent membrane. Samples and reagents were then continuously pipetted as described below. Each sample and reagent volume was overlaid on the membrane before pipetting the next volume.
[0073]
1) 30 μL of 50 mM NaPO _Four Prewash with 0.15 M NaCl, pH 7.4.
2) A filter with pre-retained biotinylated IgE was placed at the bottom of the section.
3) 30 μL of serum was pipetted to each filter.
4) 20 μL of test buffer (0.1 NaPO _Four 0.15M NaCl, 10% sucrose, 3% BSA, 0.05% bovine gamma globulin, 0.05% NaN _Three , PH 7.4) was added to the filter.
5) The allergen filter was removed.
6) 20 μl of detection conjugate diluted in test buffer (75 μg / mL).
7) 2 × 30 μL of test buffer.
8) The fluorescence in the detection zone was measured as a reaction zone (Vmm) with a scanning red laser fluorometer (635 nm).
Selected serum samples included negative, weak positive and strong positive sera.
[0074]
result
[Table 3]

[0075]
The results show that the pre-retained allergen (or antigen) in the application zone and the general binding agent in the reaction zone work well.

Claims (38)

Use biospecific affinity reactions to detect analytes in samples,
i. The sample (analyte) and the analytically detectable reactant (reactant ^* ) are passed through the flow path in the flow matrix into the matrix where there is a tightly immobilized biospecific affinity reactant (capture). Moving to the detection zone (DZ),
ii. Reactant ^* is captured in the detection zone (DZ), the amount of which is related to the amount of analyte in the sample,
Including
A flow characterized in that A) the reactants ^* have particles as analytically detectable groups, and B) the traps are immobilized on the matrix via immobilized particles and present hydrophilic groups on their surface. Methods related to analytical methods in the matrix.   The method according to claim 1, wherein the flow occurs on the side of the matrix.   3. A method according to claim 1 or 2, characterized in that the flow is caused by capillary forces.   4. A method according to any one of claims 1 to 3, characterized in that the capture body can bind to the reactant via a biospecific affinity, and then the reactant binds to the analyte biospecifically. .   The reactant is applied with the sample or pre-retained in a matrix upstream of the detection zone (DZ) so that the reactant can react with the analyte before reaching the detection zone (DZ). The method of claim 4.   6. A method according to any one of the preceding claims, characterized in that the particles fixing the capture bodies have a maximum outer dimension which is smaller than the minimum inner dimension of the matrix channel.   7. A method according to any one of claims 1 to 6, characterized in that the particles fixing the trap have a maximum outer dimension in the range of 0.1 to 1000 [mu] m.   8. A method according to claim 7, characterized in that the particles fixing the trap have a maximum outer dimension in the range of 0.1-100 [mu] m.   9. A method according to any of claims 1 to 8, characterized in that the labeled particles have a diameter of 0.01-5 [mu] m.   10. A method according to any one of the preceding claims, characterized in that the flow path has a minimum inner dimension in the range of 0.4 to 1000 [mu] m.   11. A method according to claim 10, characterized in that the flow path has a minimum inner dimension in the range of 0.4-100 [mu] m.   12. A method according to any one of the preceding claims, characterized in that the labeled particles are fluorescent or colored. 13. A method according to any one of the preceding claims, characterized in that the reactant ^* is pre-retained in the matrix upstream of the detection zone (DZ) or upstream of the sample application site in the matrix.   14. A method according to any one of the preceding claims, characterized in that the particles that immobilize the traps on the matrix are synthetic polymers or semi-synthetic polymers or biopolymers that exhibit hydrophilic groups on their surface. The measurement method is a three-component complex reactant '--- analyte --- reactor ^* (reactant' and reactant ^* can simultaneously bind to the analyte biospecifically, and reactant ' 15. A sandwich type, wherein the reactant ^* is captured in the detection zone (DZ) by the formation of a rigidly attached reactant that can bind via biospecific affinity. The method in any one of. The specimen is an antibody having specificity for either reactant 'or reactant ^*
a) When the analyte specificity of the analyte is relative to the reactant ', the reactant' is an antigen / hapten and the reactant ^* is an antibody against a constant antibody region on the analyte, or b) the antibody specificity of the analyte is the reactant 16. A method according to claim 15, characterized in that when ^{* is} directed, the reactant ^* is an antigen / hapten and the reactant 'is an antibody against a constant antibody region on the specimen. The method according to claim 15, wherein the specimen is an antigen, and the reactant 'and the reactant ^* are antibodies having specificity for an epitope on the specimen.   The method according to claim 15 or 16, characterized in that the specimen is an IgE class against allergen. The method according to any one of claims 1 to 18, characterized in that the measuring method is performed in connection with detection of allergy or autoimmune disease. Utilizing a biospecific affinity reaction for the detection of an analyte in a sample, (i) a flow matrix having a detection zone (DZ) in which a tightly immobilized biospecific affinity reactant (capture) is present; And (ii) an analytically detectable reactant (reactant ^* ),
In a flow matrix, characterized in that A) the reactants ^* have particles as analytically detectable groups, and B) the traps are immobilized on the matrix via immobilized particles and present hydrophilic groups on their surface Test kit for carrying out the analytical method.   21. Kit according to claim 20, characterized in that the flow takes place on the side of the matrix.   22. Kit according to claim 20 or 21, characterized in that the flow in the matrix is caused by capillary forces.   23. Kit according to any of claims 20 to 22, characterized in that the capture body can bind to the reactant via a biospecific affinity, which then binds the analyte to the analyte biospecifically. .   The reactant is applied with the sample or pre-retained in a matrix upstream of the detection zone (DZ) so that the reactant can react with the analyte before reaching the detection zone (DZ). The kit according to claim 23.   25. Kit according to any one of claims 20 to 24, characterized in that the particles fixing the trap have a maximum outer dimension which is smaller than the minimum inner dimension of the matrix channel.   26. Kit according to any one of claims 20 to 25, characterized in that the particles fixing the capture bodies have a maximum outer dimension in the range of 0.1 to 1000 [mu] m.   27. Kit according to claim 26, characterized in that the particles fixing the trap have a maximum outer dimension in the range of 0.1-100 [mu] m.   28. Kit according to any one of claims 20 to 27, characterized in that the labeled particles have a diameter of 0.01-5 [mu] m.   29. Kit according to any one of claims 20 to 28, characterized in that the flow path has a minimum inner dimension in the range of 0.4 to 1000 [mu] m.   30. Kit according to claim 29, characterized in that the flow path has a minimum inner dimension in the range of 0.4-100 [mu] m.   31. Kit according to any one of claims 20 to 30, characterized in that the labeled particles are fluorescent or colored. 32. Kit according to any one of claims 20 to 31, characterized in that the reactants ^* are previously retained in the matrix upstream of the detection zone (DZ) or upstream of the sample application site in the matrix.   The kit according to any one of claims 20 to 32, wherein the particles for immobilizing the capturing body on the matrix are a synthetic polymer, a semi-synthetic polymer or a biopolymer that exhibits a hydrophilic group on the surface thereof. The kit is a three-component complex reactant '--- analyte --- reactor ^* (reactant' and reactant ^* can simultaneously bind to the analyte biospecifically, and the reactant ' Characterized in that it is intended for a sandwich-type assay that captures the reactant ^* in the detection zone (DZ) by the formation of a tightly bound reactant that can bind via specific affinity) The kit according to any one of claims 20 to 33. The specimen is an antibody having specificity for either reactant 'or reactant ^*
a) When the analyte specificity of the analyte is relative to the reactant ', the reactant' is an antigen / hapten and the reactant ^* is an antibody against a constant antibody region on the analyte, or b) the antibody specificity of the analyte is the reactant ^* when for the reactants ^* is an antigen / hapten, and wherein the reactants' is an antibody to the constant antibody regions on the specimen, according to claim 34 kit according. The kit according to claim 34, wherein the specimen is an antigen, and the reactant 'and the reactant ^* are antibodies having specificity for an epitope on the specimen.   36. Kit according to claim 34 or 35, characterized in that the specimen is an IgE class against allergen.   The kit according to any one of claims 20 to 37, wherein the measuring method is performed in connection with diagnosis of allergy or autoimmune disease.